Identification of fetal aneuploidy with dual-probe fluorescence in situ hybridization analysis in circulating trophoblasts after enrichment using a high-sensitivity microfluidic platform.

OBJECTIVE: To evaluate a microfluidics-based positive selection technology for isolating circulating trophoblasts (CTs) from peripheral blood of women whose pregnancies are affected by aneuploidy and to evaluate fetal karyotype using fluorescence in situ hybridization (FISH). METHOD: Ten 18-ml samples of peripheral blood were collected consecutively from pregnant women whose fetus was affected by aneuploidy. A preservation buffer was added, and the specimens were shipped overnight to the testing laboratory at ambient temperature. The specimen was infused into the fully automated microfluidics-based LiquidScan® instrument without pre-processing. This instrument contains microfluidic chips, which are coated with antibodies (anti-huEpCAM and a proprietary antibody mixture) specific to CT surface epitopes. FISH analysis was performed on the enriched cells. RESULTS: Fetal aneuploidy evaluated included trisomy 21 (n = 3), trisomy 18 (n = 1), trisomy 13 (n = 1), monosomy X (n = 3), and triploidy (n = 1). CTs for analysis by FISH were identified in all samples. The average number of mononucleate cells per 1 ml of whole blood was 2.11 (range 0.38-4.63) overall and was 2.67 (range 1.13-4.63) using the proprietary combination of antibodies. FISH results were concordant with the aneuploidy based on other testing in all cases. Multinucleate cells were searched for and identified in the last seven samples (average number: 0.84/1 ml). CONCLUSIONS: Our study demonstrates that the LiquidScan® , a high-sensitivity microfluidic platform, can enrich circulating trophoblasts (mononucleate and multinucleate). FISH can then be used to detect fetal aneuploidy.

The dependence of the optoelectrical properties of silver nanowire networks on nanowire length and diameter.

We have characterized the optoelectrical properties of networks of silver nanowires as a function of nanowire dimension by measuring transmittance (T) and sheet resistance (R(s)) for a large number of networks of different thicknesses fabricated from wires of different diameters (D) and lengths (L). We have analysed these data using both bulk-like and percolative models. We find the network DC conductivity to scale linearly with wire length while the optical conductivity is approximately invariant with nanowire length. The ratio of DC to optical conductivity, often taken as a figure of merit for transparent conductors, scales approximately as L/D. Interestingly, the percolative exponent, n, scales empirically as D², while the percolative figure of merit, Π, displays large values at low D. As high T and low R(s) are associated with low n and high Π, these data are consistent with improved optoelectrical performance for networks of low-D wires. We predict that networks of wires with D = 25 nm could give sheet resistance as low as 25 Ω/□ for T = 90%.

Microfluidic affinity selection of active SARS-CoV-2 virus particles.

We report a microfluidic assay to select active severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles (VPs), which were defined as intact particles with an accessible angiotensin-converting enzyme 2 receptor binding domain (RBD) on the spike (S) protein, from clinical samples. Affinity selection of SARS-CoV-2 particles was carried out using injection molded microfluidic chips, which allow for high-scale production to accommodate large-scale screening. The microfluidic contained a surface-bound aptamer directed against the virus's S protein RBD to affinity select SARS-CoV-2 VPs. Following selection (~94% recovery), the VPs were released from the chip's surface using a blue light light-emitting diode (89% efficiency). Selected SARS-CoV-2 VP enumeration was carried out using reverse transcription quantitative polymerase chain reaction. The VP selection assay successfully identified healthy donors (clinical specificity = 100%) and 19 of 20 patients with coronavirus disease 2019 (COVID-19) (95% sensitivity). In 15 patients with COVID-19, the presence of active SARS-CoV-2 VPs was found. The chip can be reprogrammed for any VP or exosomes by simply changing the affinity agent.

The threshold length for fiber-induced acute pleural inflammation: shedding light on the early events in asbestos-induced mesothelioma.

Suspicion has been raised that high aspect ratio nanoparticles or nanofibers might possess asbestos-like pathogenicity. The pleural space is a specific target for disease in individuals exposed to asbestos and by implication of nanofibers. Pleural effects of fibers depends on fiber length, but the key threshold length beyond which adverse effects occur has never been identified till now because all asbestos and vitreous fiber samples are heterogeneously distributed in their length. Nanotechnology advantageously allows for highly defined length distribution of synthetically engineered fibers that enable for in-depth investigation of this threshold length. We utilized the ability to prepare silver nanofibers of five defined length classes to demonstrate a threshold fiber length for acute pleural inflammation. Nickel nanofibers and carbon nanotubes were then used to strengthen the relationship between fiber length and pleural inflammation. A method of intrapleural injection of nanofibers in female C57Bl/6 strain mice was used to deliver the fiber dose, and we then assessed the acute pleural inflammatory response. Chest wall sections were examined by light and scanning electron microscopy to identify areas of lesion; furthermore, cell-nanowires interaction on the mesothelial surface of the parietal pleura in vivo was investigated. Our results showed a clear threshold effect, demonstrating that fibers beyond 4 µm in length are pathogenic to the pleura. The identification of the threshold length for nanofiber-induced pathogenicity in the pleura has important implications for understanding the structure-toxicity relationship for asbestos-induced mesothelioma and consequent risk assessment with the aim to contribute to the engineering of synthetic nanofibers by the adoption of a benign-by-design approach.

Post-tobacco master settlement agreement: policy and practice implications for social workers.

The 1998 Tobacco Master Settlement Agreement (MSA) was developed between states and tobacco manufacturers to settle the states' lawsuits against tobacco manufacturers and recover tobacco health-related costs. States won billions of dollars and concessions regarding how tobacco products could be advertised. The purpose of the MSA was to prevent cigarette smoking and compensate for health expenses incurred in the treatment of tobacco smoking-related illnesses. Twelve years after the settlement, it is clear that MSA monies have been gravely diverted from tobacco prevention and cessation programs to balance budgets. The authors' review indicates that increases in funding for state tobacco prevention and cessation programs reduce tobacco use across all populations. Implications for social policy and social work practice are offered.

Enzyme-mediated individual nanoparticle release assay.

Numerous methods have been developed to measure the presence of macromolecular species in a sample; however, the number of methods that detect functional activity or modulators of that activity is more limited. To address this limitation, an approach was developed that uses the optical detection of nanoparticles as a measure of enzyme activity. Nanoparticles are increasingly being used as biological labels in static binding assays; here, we describe their use in a release assay format, where the enzyme-mediated liberation of individual nanoparticles from a surface is measured. A double-stranded fragment of DNA is used as the initial tether to bind the nanoparticles to a solid surface. The nanoparticle spatial distribution and number are determined using dark-field optical microscopy and digital image capture. Site-specific cleavage of the DNA tether results in nanoparticle release. The methodology and validation of this approach for measuring enzyme-mediated, individual DNA cleavage events, rapidly, with high specificity, and in real-time are described. This approach was used to detect and discriminate between nonmethylated and methylated DNA, and demonstrates a novel platform for high-throughput screening of modulators of enzyme activity.

Peptidoglycan recognition protein expression in mouse Peyer's Patch follicle associated epithelium suggests functional specialization.

Mammalian Peyer's Patches possess specialized epithelium, the follicle associated epithelium (FAE), and specialized cells called M cells which mediate transcytosis of antigens to underlying lymphoid tissue. To identify FAE specific genes, we used TOGA gene expression profiling of microdissected mouse Peyer's Patch tissue. We found expression of laminin beta3 across the FAE, and scattered expression of peptidoglycan recognition protein (PGRP)-S. Using the M cell specific lectin Ulex europaeus agglutinin 1 (UEA-1), PGRP-S expression was nearly exclusively co-localized with UEA-1+ M cells. By contrast, the related gene PGRP-L was expressed among a subset of UEA-1 negative FAE cells. Expression of these proteins in transfected cells demonstrated distinct subcellular localization. PGRP-S showed a vesicular pattern and extracellular secretion, while PGRP-L showed localization to both the cytoplasm and the cell surface. The potential function of these PGRP proteins as pattern recognition receptors and their distinctive cellular distribution suggests a complex coordination among specialized cells of the FAE in triggering mucosal immunity and innate immune responses.

Cell culture modeling of specialized tissue: identification of genes expressed specifically by follicle-associated epithelium of Peyer's patch by expression profiling of Caco-2/Raji co-cultures.

Peyer's patch follicle-associated epithelium (FAE) regulates intestinal antigen access to the immune system in part through the action of microfold (M) cells which mediate transcytosis of antigens and microorganisms. Studies on M cells have been limited by the difficulties in isolating purified cells, so we applied TOGA mRNA expression profiling to identify genes associated with the in vitro induction of M cell-like features in Caco-2 cells and tested them against normal Peyer's patch tissue for their expression in FAE. Among the genes identified by this method, laminin beta3, a matrix metalloproteinase and a tetraspan family member, showed enriched expression in FAE of mouse Peyer's patches. Moreover, the C. perfringens enterotoxin receptor (CPE-R) appeared to be expressed more strongly by UEA-1(+) M cells relative to neighboring FAE. Expression of the tetraspan TM4SF3 gene and CPE-R was also confirmed in human Peyer's patch FAE. Our results suggest that while the Caco-2 differentiation model is associated with some functional features of M cells, the genes induced may instead reflect the acquisition of a more general FAE phenotype, sharing only select features with the M cell subset.